PROJECT SUMMARY/ABSTRACT The trophectoderm layer of the blastocyst-stage embryo is the precursor of all trophoblast cell types in the pla- centa. Abnormalities in early trophoblast development are associated with adverse maternal and fetal health outcomes. Yet, despite its significance, early human trophoblast development remains poorly understood due to constraints on research with human embryos and limited availability of fetal tissue from early gestation. The central hypothesis of this proposal is that human trophoblast stem cells (hTSCs) derived from human in- duced pluripotent stem cells (hiPSCs) can be used to accurately model early human placental develop- ment in vitro. Specifically, we will generate hTSCs using hiPSCs that were derived from umbilical cords of normal pregnancies and those associated with early onset preeclampsia (EOPE). Differentiation of epithelial trophoblast cells to extravillous trophoblasts (EVTs), and subsequent invasion of uterine tissue by a subset of EVTs that have a mesenchymal phenotype, is a vital process in early human placental development. We will use a novel 3D cell culture system to investigate differences in invasion of EVTs obtained from normal and EOPE-associated hTSCs. Two specific aims are proposed. In Aim 1, we will derive hTSCs using hiPSCs from umbilical cords of normal pregnancies and those associated with EOPE. We will validate the self-renewal capa- bility of hTSCs and their potential to differentiate into EVTs and syncytiotrophoblast using previously described protocols. In Aim 2, we will investigate quantitative differences in trophoblast differentiation and invasion be- tween normal and EOPE-associated hTSCs. We will use a novel 3D cell culture system and a chemically defined medium for these studies. The lack of an in vitro cell culture system from non-fetal sources that accurately models human trophoblast development is a formidable barrier for the field of human placental biology. Our proposed research is significant because hiPSC-derived hTSCs provide a unique and powerful model system based on non-fetal sources. Specifically, the use of hiPSC-derived hTSCs provides two important advantages. First, the ease of deriving hiPSCs ? relative to using human placental samples from early gestation ? will enable facile derivation of hTSCs from normal and pathological pregnancy outcomes. Second, hiPSC-derived hTSCs over- come an important limitation encountered when using early gestation placental samples or hTSCs derived from such samples. In contrast to primary samples where the projected health outcome of an elective termination of pregnancy at term is difficult to ascertain or confirm, the use of hiPSCs from umbilical cords at full term allow development of hTSC models for normal placental development.